Method for treatment of hemophilia by extravascular administration of factor VIII deletion derivatives

ABSTRACT

A pharmaceutical formulation for subcutaneous, intramuscular or intradermal administration comprising recombinant coagulation factor VIII and use thereof for manufacture of a medicament for treating haemophilia is provided. The formulation comprises a highly purified recombinant coagulation factor VIII in a concentration of at least 1000 IU/ml, which gives surprisingly high levels of active factor VIII in the blood stream after subcutaneous, intramuscular or intradermal administration. The formulation is intended for treatment of haemophilia by subcutaneous, intramuscular or intradermal administration. The recombinant factor VIII is preferably a deletion derivative thereof, which can be used for the manufacture of a medicament for subcutaneous administration.

This application claims the benefit of Swedish Application No.9302308-3, filed on Jul. 5, 1993, and PCT/SE94/00297, filed Mar. 31,1994.

The present invention relates to a pharmaceutical formulation forsubcutaneous, intramuscular or intradermal administration comprisingrecombinant coagulation factor VIII and use thereof for manufacture of amedicament for treating haemophilia. The formulation comprises a highlypurified recombinant coagulation factor VIII in a concentration of atleast 1000 IU/ml, which gives surprisingly high levels of active factorVIII in the blood stream after subcutaneous, intramuscular orintradermal administration. The formulation is intended for treatment ofhaemophilia by subcutaneous, intramuscular or intradermaladministration. The recombinant factor VIII is preferably a deletionderivative thereof, which can be used for the manufacture of amedicament for subcutaneous administration.

BACKGROUND OF THE INVENTION

Haemophilia is an inherited disease which has been known for centuriesbut it is only within the last three decades that it has been possibleto differentiate between the various forms; haemophilia A, haemophilia Band haemophilia C. Haemophilia A is the most frequent form. It affectsonly males with an incidence of one or two individuals per 10 000live-born males. The disease is caused by strongly decreased level orabsence of biologically active coagulation factor VIII (antihaemophilicfactor), which is a protein normally present in plasma. The clinicalmanifestation of haemophilia A is a strong bleeding tendency and beforetreatment with factor VIII concentrates was introduced, the mean age ofthose patients was less than 20 years. Concentrates of factor VIIIobtained from plasma have been available for about three decades. Thishas improved the situation for treatment of haemophilia patientsconsiderably and given them possibility to live a normal life.

Therapeutic factor VIII concentrates have until now been prepared byfractionation of plasma. However, there are now methods available forproduction of factor VIII in cell culture using recombinant DNAtechniques as reported in e.g. J Gitschier et al. Nature 312, p.330-37,1984 and EP-A-160 457.

Factor VIII concentrates derived from human plasma contain severalfragmented fully active factor VIII forms (Andersson et al, Proc. Natl.Acad. Sci. USA, Vol 83, p. 2979-83, May 1986). The smallest active formhas a molecular mass of 170 kDa and consists of two chains of 90 kDa and80 kDa held together by a metal ion bridge. Reference is here made toEP-A-197 901. Kabi Pharmacia has developed a recombinant factor VIIIproduct which corresponds to the 170 kDa plasma factor VIII form intherapeutic factor VIII concentrates. The truncated recombinant factorVIII molecule is termed r-VIII SQ and is produced by Chinese HamsterOvary (CHO) cells in a cell culture process in serum-free medium.

The structure and biochemistry of recombinant factor VIII products ingeneral have been described by Kaufman in Tibtech, Vol 9, 1991 andHematology, 63, p. 155-65, 1991. The structure and biochemistry ofr-VIII SQ have been described in WO-A-91/09122.

Large proteins are normally given intravenously so the medicamentdirectly available in the blood stream. It would however be advantageousif a medicament could be given subcutaneously, intramuscularly orintradermally as these administration forms are much easier to handlefor the patient. Especially if the medicament must be taken regularlyduring the whole life and treatment is to start early, already when thepatient is a child. However, a medicament with a very large and labilemolecule, such as coagulations factor VIII of 170 to 300 kDa, havenormally a very low bioaviability if given subcutaneously,intramuscularly or intradermally, since the uptake is not enough anddegradation is severe. To our knowledge, the only coagulation factorprotein which has been administered by subcutaneous injection is factorIX (90 kDa).

All presently available factor VIII preparations on the market are madeas a formulation for intravenous administration and are stabilised withhuman serum albumin.

DESCRIPTION OF THE INVENTION

To our great surprise we have found that factor VIII, which is a verysensitive protein, can be given subcutaneously and, in contrast to allearlier experience we obtain an acceptable absorption and a high levelof active factor VIII protein in the blood.

We have thus developed a formulation which makes it possible toadminister factor VIII subcutaneously, intramuscularly or intradermallyand which gives a great advantage for all patients in need of factorVIII.

Recombinant factor VIII SQ is indicated for treatment of classicalhaemophilia. The half-life for r-VIII SQ is approximately 12 hours forhumans when injected intravenously. For prophylactic treatment 15-25IU/kg bodyweigt is given of factor VIII three times a week. Anintravenous injection is normally 5-10 ml. An injection givensubcutaneously is between 0.05 to 1 ml and the concentration of factorVIII must therefore be very high in such a formulation. This is possibleto obtain e.g. with our highly purified recombinant factor VIII.

The inventive idea is thus a combination of the finding that factor VIIIcan be absorbed into the blood-stream when given as a subcutaneous,intramuscular or intradermal pharmaceutical formulation and that it ispossible to produce a formulation comprising the required highconcentration of factor VIII for this purpose.

The present invention relates to a pharmaceutical formulation forsubcutaneous, intramuscular or intradermal administration comprisinghighly purified recombinant coagulation factor VIII in a concentrationof at least 1000 IU/ml, which formulation gives a therapeutic level offactor VIII activity in the blood after administration.

The composition is preferably given subcutaneously. The factor VIIIactivity in the formulation is at least 1000 IU/ml, preferably more than1500 IU/ml and most preferably from 5000 to 100 000 IU/ml. The volumegiven is suitably 0.1 to 2 ml, preferably 0.25 to 1.5 ml, and morepreferably 0.5 to 1 ml. The volume can also be 0.1 to 1 ml. Factor VIIIis recombinant and it can be either in its full-length form orpreferably a deletion derivative thereof. More preferably the deletionderivative is recombinant factor VIII SQ (r-VIII SQ). By deletionderivative is here meant coagulation factor VIII, in which the whole orpart of the B-domain is missing. Additionally, the r-VIII SQ moleculecan be chemically modified, e.g. by pegylation, covalently linedcarbohydrates or polypeptides, in order to improve the stability of themolecule in vivo.

Our used factor VIII is highly purified, i.e. has a specific activity ofmore than 5000 IU/mg protein, even more than 12 000 IU/mg and ispreferably stabilized without the addition of albumin.

The formulation can also comprise sodium or potassium chloride,preferably in an amount of more than 0.1 M.

Calcium (or other divalent metal ions) is necessary for the maintenanceof the association of factor VIII heavy and light chain. It is hereadded as calcium chloride (CaCl₂) but other salts such as calciumgluconate, calcium glubionate or calcium gluceptate can also be used.The composition comprises preferably calcium chloride or calciumgluconate in an amount of more than 0.5 mM.

An amino acid is preferably used to buffer the system and it alsoprotects the protein in the amorphous phase if the formulation isfreeze-dried. A suitable buffer could be L-histidine, lysine and/orarginine. L-Histidine has primarily been chosen because of the goodbuffer capacity of L-histidine around pH 7.

The formulation could comprise

i) at least 1500 IU/ml of a deletion derivative of recombinant factorVIII

ii) at least 0.01 mg/ml of a polyoxyethylene sorbitan fatty acid ester

iii) sodium chloride, preferably in an amount of more than 0.1 M.

iv) calcium salt, such as calcium chloride or calcium gluconate,preferably in an amount of more than 0.5 mM.

v) an amino acid such as L-histidine in an amount of more than 1 mM.

A non-ionic surfactant can also be present in the formulation and isthen preferably chosen from block co-polymers, such as a poloxamer orpolyoxyethylene sorbitan fatty acid ester, such aspolyoxyethylene-(20)-sorbitan monolaurate orpolyoxyethylene-(20)-sorbitan monooleate. The non-ionic surfactant, ifused, should preferably be present in an amount above the criticalmicelle concentration (CMC). See Wan and Lee, Journal of Pharm Sci, 63,p.136, 1974. The polyoxyethylene sorbitan fatty acid ester is preferablyused in an amount of at least 0.01 mg/ml.

To this formulation mono- or disaccharides or sugar alcohols, preferablysucrose, could be added. Also antioxidants such as gluthatione,acetylcystein, tocopherol, methionin, EDTA, citric acid, butyl hydroxytoluene and/or butyl hydroxy anisole could be added. Furthermore,preservatives such as benzyl alcohol, phenol, sorbic acid, parabens andchlorocresol could be added.

The formulation comprises preferably L-histidine and sucrose. The ratioof sodium chloride to L-histidine and sucrose in the composition forfreeze-drying is suitably more than 1:1 (w:w), preferably more than 2:1(w:w).

The formulation could be in a dried form, preferably freeze-dried. Thedried product is reconstituted with sterile water for injection or apharmaceutically acceptable buffer solution, or mixed or reconstitutedwith an aqueous solution containing absorption enhancers or proteaseinhibitors before administration.

The claimed formulation can also be a stable aqueous solution ready foradministration. It can also be a dispersion, e.g. a suspension, aliposomal formulation or an emulsion.

The formulation could be stored in an oxygen-reduced environment asdisclosed in the copending patent application PCT/SE94/00265.

Absorption enhancers or protease inhibitors could be added. Examples ofsuitable absorption enhancers are phospholipids, fatty acids, bilesalts, salicylates and EDTA. Examples of suitable protease inhibitorsare aprotinin and EDTA.

The claimed formulation can be prepared by mixing factor VIII with anon-ionic surfactant in an aqueous solution, preferably together with anamino acid such as L-histidine, sodium salt, sucrose and a calcium saltor by eluating factor VIII from the last purification step with a buffercontaining a non-ionic surfactant in an aqueous solution, preferablytogether with sodium salt, sucrose, calcium salt and an amino acid suchas L-histidine.

The invention also relates to the use of the claimed formulation for themanufacture of a medicament for subcutaneous, intramuscular orintradermal administration for treating haemophilia, preferably for theuse of a deletion derivative of recombinant factor VIII for themanufacture of a medicament for subcutaneous administration. Themedicament can be in a stable aqueous solution or dispersion, orfreeze-dried. It also relates to a method for treatment of haemophiliaby subcutaneous, intramuscular or intradermal administration of theclaimed formulation.

The data presented in the examples indicate that r-VIII SQ can beinjected subcutaneously and recovered in an active form intravenously invivo. This is a very surprising finding, as no such formulation havebeen known earlier.

The protection is not limited to a composition under these examples.

EXPERIMENTAL Material and Methods

The production of recombinant factor VIII SQ(r-VIII SQ) was essentiallyperformed as described in patent WO-A-91/09122, example 1-3. A DHFRdeficient CHO celline (DG44N.Y.) was electroporated with an expressionvector containing the r-VIII SQ gene and an expression vector containingthe dihydroflolate-reductase gene. Following selection on selectivemedia surviving colonies were amplified through growth in stepwiseincreasing amounts of methotrexate. Supernatant from the resultingcolonies were individually screened for factor VIII activity. Aproduction clone was chosen and this was subsequently adapted to serumfree suspension growth in a defined medium and finally a large scalefermentation process was developed. Supernantant is collected aftercertain time periods and further purified as described below.

The clarified conditioned medium was pH adjusted and applied to aS-Sepharose FF column. After washing, factor VIII was eluated with asalt buffer containing 5 mM CaCl₂.

Immunoadsorption was carried out on an immunoaffinity resin where theligand was a monoclonal antibody (8A4) directed towards the heavy chainof factor VIII. Before loading to the column the S-eluate was treatedwith 0.3% TNBP and 1% Octoxynol 9.

The column was equilibrated, washed and factor VIII was eluated with abuffer containing 0.05 M CaCl₂ and 50% ethylene glycol.

The mAb-eluate was loaded on a Q-Sepharose FF column, equilibrated withthe elution buffer in the immunoaffinity step. After washing, factorVIII was eluated with 0.05 M L-Histidine, 0.6 M sodium chloride, 4 mMcalcium chloride and pH 6.8.

The Q-eluate was applied to a gel filtration column (Superdex 200 p.g.).Equilibration and elution was carried out with a buffer containingL-histidine, sodium chloride and calcium chloride. The protein peak wascollected and the solution was formulated before freeze-drying.

This material of r-VIII SQ was received from the final purificationstep. The factor VIII activity and the concentration of the inactivecomponents were adjusted by diluting with an appropriate buffercontaining polyethylene glycol (PEG). The solution was then sterilefiltered (0.22 μm), dispensed and freeze-dried.

EXAMPLE 1

Recombinant factor VIII was prepared according to the method describedunder Experimental.

The freeze-dried composition containing r-VIII SQ was the following pervial, which was reconstituted in 4 ml sterile water for injection:

    ______________________________________                                        Composition per vial:                                                         ______________________________________                                        L-Histidine, mg       31.0                                                    Sodium chloride, mg   70.1                                                    Calcium chloride.(2H.sub.2 O), mg                                                                    2.35                                                   Polyethylene glycol (PEG 4000), mg                                                                   4.0                                                    Polyoxyethylene-(20)-sorbitan                                                                         1                                                     monooleate (Tween 80 ®), mg                                               VIII:C charged, IU/vial                                                                             4400                                                    VIII:C in reconstituted solution, IU/ml                                                             1060                                                    ______________________________________                                    

Male albino mice weighing about 30 g and of strain NMRI, SPF, wereinjected subcutaneously in the neck with the reconstituted r-VIII Sqsolution. The volume injected at the dose level of 10 000 IU/kg was 9.4ml/kg and at the higher dose level 50 000 IU/kg, 5 times larger, 47ml/kg. In the placebo treatment saline was used, 9.4 ml/kg. 3-5 minutesbefore the blood sampling the mice were anaesthetized intra peritoneallywith Mebumal® (pentobarbital) vet. 60 mg/ml. The volume injected was 9.4ml/kg i.e. about 0.3 ml/mouse. Under the anaesthesia 0.45 ml blood wascollected from the vena cava in plastic syringes containing 0.05 ml 0.13M sodium citrate. Plasma was then prepared from the collected blood bycentrifugation (8800 g for 7 minutes) and kept frozen in plasticCryoflex tubes at -70° C. until the time of determination of factor VIIIactivity.

Results:

                  TABLE 1                                                         ______________________________________                                        VIII:C in plasma from mice receiving r-VIII SQ 10000 IU/kg                    body weight subcutaneously.                                                   Time after      VIII:C    n                                                   administration  (IU/ml)   number of                                           (hours)         X ± Sd observations                                        ______________________________________                                        0               0.82 ± 0.43                                                                          12                                                  0.33            1.23 ± 0.41                                                                          3                                                   1.0             1.38 ± 0.56                                                                          4                                                   1.5             1.83 ± 0.60                                                                          12                                                  2.0             1.43 ± 0.91                                                                          8                                                   4.0             1.34 ± 0.72                                                                          6                                                   6.0             1.27 ± 0.42                                                                          4                                                   8.0             1.55 ± 0.80                                                                          4                                                   16.0            0.62 ± 0.28                                                                          4                                                   20.0            0.58 ± 0.12                                                                          3                                                   24.0            0.76 ± 0.53                                                                          4                                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Dose - Response in mice receiving r-VIII SQ subcutaneosly.                    Dose: Dose r-VIII SQ administrated (IU/kg).                                   Response: VIII:C in plasma (IU/ml) 1.5 hours after subcutaneous               administration.                                                                                     Response                                                                      (IU/ml)                                                 Dose     Response     (baseline                                                                              n                                              (IU/kg)  (IU/ml)      adjusted)                                                                              number of obs.                                 ______________________________________                                        Blank    0.82 ± 0.43                                                                             --       12                                             (baseline)                                                                    Saline   1.21 ± 0.34                                                                             0.39     4                                              10000    1.83 ± 0.60                                                                             1.01     12                                             50000    2.47 ± 0.60                                                                             1.65     6                                              ______________________________________                                    

Results

1. The change of VIII:C in plasma with time after a subcutaneous dose ofr-VIII SQ, 10 000 IU/kg, shows the typical pattern for a drug beingabsorbed from a subcutaneous depot (see Table 1). The maximum VIII:Clevel in plasma is seen at about 1.5 hours after administration (seeTable 1).

2. The absorption of r-VIII SQ from a subcutaneous depot is furtherverified by the increase in maximum concentration seen when the dose isincreased five-fold. No significant effect was observed on the obtainedplasma level of VIII:C when the subcutaneous injection volume waschanged from 9.4 to 47 ml/kg while the dose of r-VIII Sq was keptconstant. Furthermore, the VIII:C obtained in plasma was essentially notdependent on the osmolality of the administration solution.

3. The bioavailability of r-VIII SQ after subcutaneous administration inmouse was about 10% of the bioavailability after intravenousadministration. The bioavailability was calculated from the area underthe activity (VIII:C)--time curve.

EXAMPLE 2

Plasma derived factor VIII was used.

The composition of the frozen solution containing plasma derived factorVIII was the following:

    ______________________________________                                        Composition per ml:                                                           ______________________________________                                        L-Histidine, mg       7.8                                                     Sodium chloride, mg   35.1                                                    Calcium chloride.(2H.sub.2 O), mg                                                                    0.59                                                   Polyethylene glycol (PEG 4000), mg                                                                  1.0                                                     Albumin human, mg     10.0                                                    VIII:C, IU/ml         250                                                     ______________________________________                                    

Male albino mice weighing about 30 g and of strain NMRI, SPF, wereinjected subcutaneously in the neck with the reconstituted factor VIIIsolution.

The volume injected at the dose level of 10 000 IU/kg was about 40ml/kg. In the placebo treatment a solution containing 9% (w/v) sodiumchloride was used. 3-5 minutes before the blood sampling the mice wereanaesthetized intra peritoneally with Mebumal® (pentobarbital) vet. 60mg/ml. The dose volume was about 1.2 ml/mouse. Under anasthesia 0.45 mlblood was collected from vena cava in plastic syringes containing 0.05ml sodium citrate (0.13 M). Plasma was prepared from the blood bycentrifugation (8800 g for 7 minutes) and thereafter stored in plasticCryoflex tubes at -70 ° C. until determination of VIII:C.

Results:

                  TABLE 3                                                         ______________________________________                                        VIII:C in plasma from mice receiving plasma derived factor VIII,              10000 IU/kg, subcutaneously. VIII:C in the administration                     solution was about 250 IU/ml.                                                 Time after                                                                    administration                                                                            VIII:C (IU/ml)                                                                           n                                                      (hours)     X ± SD  number of observations                                 ______________________________________                                        0           1.04 ± 0.30                                                                           4                                                      1           1.20 ± 0.53                                                                           4                                                      1.5         1.39 ± 0.04                                                                           4                                                      2           0.85 ± 0.34                                                                           4                                                      4           1.19 ± 0.49                                                                           4                                                      6           1.32 ± 0.53                                                                           4                                                      8           1.15 ± 0.39                                                                           4                                                      16          1.27 ± 0.08                                                                           4                                                      20          1.17 ± 0.41                                                                           4                                                      24          1.32 ± 0.21                                                                           4                                                      ______________________________________                                    

Results:

1. The bioavailability of plasma derived factor VIII is fairly lowaccording to the results of the in vivo study in mice (Table 3). Thebioavailability of plasma derived factor VIII is roughly about 1/10 ofthe bioavailability of r-VIII SQ. Hence, the bioavailability of r-VIIISQ is substantially higher than the bioavailability of factor VIIIderived from human plasma. The maximum plasma level of VIII:C is seenabout 1.5 hours after subcutaneous administration.

EXAMPLE 3

Recombinant factor VIII was prepared according to the method describedunder Experimental with the following exceptions: (i) the material ofr-VIII SQ that was received from the final purification step was dilutedwith a buffer not containing PEG, (ii) the r-VIII SQ solution was notfreeze-dried, it was stored at -70° C.

The r-VIII SQ-solution had the following composition:

    ______________________________________                                        Composition per vial:                                                         ______________________________________                                        L-Histidine, mg        7.5                                                    Sucrose, mg             158                                                   Sodium chloride, mg     45                                                    Calcium chloride.(2H.sub.2 O), mg                                                                    1.25                                                   Polyoxyethylene-(20)-sorbitan monooleate                                                             0.50                                                   (Tween 80 ®, mg)                                                          VIII:C charged, IU/vial                                                                              6070                                                   VIII:C, IU/ml*         1130                                                   ______________________________________                                         *Diluted rVIII SQsolution (1 part of rVIII SQsolution + 1 part of water,      v:v)                                                                     

The r-VIII SQ-solution was diluted with sterile water for injection (1part of r-VIII SQ-solution+1 part of water, v:v) prior to administrationwhen the dose of VIII:C was below 3000 IU/kg body-weight.

Female cynomolgus monkey (Macaca fascicularis) weighing about 3-3.5 kgwere injected with the r-VIII SQ-solution subcutaneously in the dorsalregion. Depending on the dose, the volume of injection was variedbetween approximately 0.2 to 2.0 ml/kg body-weight. Subcutaneousinjections with single doses of 250, 2500 and 5000 IU/kg wereadministered. On each sampling occasion 1.8 ml of blood was collectedinto tubes containing citrate as anticoagulant (0.2 ml). Aftercentrifugation plasma was separated and frozen in aliquots (<-60° C.).

Results:

                  TABLE 4                                                         ______________________________________                                        VIII:C in plasma from monkeys receiving r-VIII SQ, 250 IU/kg,                 subcutaneously. The VIII:C in the administration solution was                 about 1130 IU/ml.                                                                      Monkey No                                                            Sampling   1            2       3                                             after inj. VIII:C       VIII:C  VIII:C                                        (Hours)    (IU/ml)      (IU/ml) (IU/ml)                                       ______________________________________                                         0         2.15         1.49    1.76                                           1         2.32         1.75    1.92                                           4         2.43         1.52    1.89                                           8         2.48         1.67    1.92                                          10         2.41         1.76    2.03                                          12         2.29         1.70    2.01                                          14         2.20         1.69    2.00                                          24         1.76         1.32    1.98                                          30         2.31         1.46    1.90                                          48         2.18         1.64    1.96                                          ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        VIII:C in plasma from monkeys receiving r-VIII SQ, 2500 IU/kg,                subcutaneously. The VIII:C in the administration solution was                 about 1130 IU/ml.                                                                      Monkey No                                                            Sampling   4            5       6                                             after inj. VIII:C       VIII:C  VIII:C                                        (Hours)    (IU/ml)      (IU/ml) (IU/ml)                                       ______________________________________                                         0         1.95         1.35    2.30                                           2         2.43         1.86    3.05                                           6         3.51         2.45    3.59                                           9         3.91         2.47    4.16                                          11         3.47         2.24    3.68                                          13         3.11         2.06    3.34                                          22         2.38         1.55    2.79                                          40         2.01         1.49    2.38                                          ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        VIII:C in plasma from monkeys receiving r-VIII SQ, 5000 IU/kg,                subcutaneously. The VIII:C in the administration solution was                 about 2470 IU/ml.                                                                      Monkey No                                                            Sampling   7            8       9                                             after inj. VIII:C       VIII:C  VIII:C                                        (Hours)    (IU/ml)      (IU/ml) (IU/ml)                                       ______________________________________                                         0         2.04         1.78    1.54                                           2         2.99         2.37    2.99                                           6         4.56         3.33    5.32                                           9         4.75         3.89    5.70                                          11         4.72         3.82    5.46                                          13         4.23         3.06    4.63                                          22         3.03         2.33    3.46                                          40         2.76         1.77    2.60                                          ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Dose - Response in monkey receiving r-VIII SQ subcutaneously.                 Dose: Dose r-VIII SQ administrated (IU/kg bodyweight).                        Response: VIII:C in plasma (IU/ml) about 9 hours after                        administration.                                                                                     Response  n                                             Dose     Response     (IU/ml,   number of                                     (IU/kg)  (IU/ml)      baseline adj.)                                                                          observations                                  ______________________________________                                        Blank    1.8 ± 0.3 --        9                                             (baseline)                                                                     250                  0.37 ± 0.05                                                                          3                                             2500                  1.70 ± 0.06                                                                          3                                             2500                  2.05 ± 0.57                                                                          3                                             5000                   3.4 ± 1.20                                                                          3                                             ______________________________________                                    

Results

1. According to the results (table 4-6) the plasma concentration as afunction of time follows the typical pattern for a drug being absorbed.after subcutaneous administration. The maximum concentration of VIII:Cis seen about 9 hours after administration.

2. The dose-response relationship obtained (table 7), gives a furtherverification that there is an absorption of VIII:C into the blood streamfollowing a subcutaneous injection.

3. The bioavailability of r-VIII Sq after subcutaneous administration inmonkey was about 5-10%. The bioavailability was calculated from the areaunder the activity (VIII:C)--time curve. The bioavailability wasessentially independent of the dose of r-VIII SQ,

What is claimed is:
 1. Method for treatment of haemophilia bysubcutaneous, intramuscular or intradermal administration ofpharmaceutical formulation with a volume of 0.1 to 2 ml comprising ahighly purified recombinant coagulation factor VIII with an activity ofat least 1,000 IU/ml and a non-ionic surfactant, wherein saidrecombinant coagulation factor VIII is selected from the groupconsisting of deletion derivatives of factor VIII where the whole orpart of the B-domain is missing and factor VIII which has beenchemically modified by pegylation or covalently linked carbohydrates orpolypeptides, which formulation is stabilized without albumin and iscapable of increasing the factor VIII activity in the blood to atherapeutic level after subcutaneous, intramuscular or intradermaladministration.
 2. A method according to claim 1 in which the factorVIII activity is from 5000 to 100000 IU/ml.
 3. A method according toclaim 1 wherein the formulation has a volume of 0.5 to 1 ml.
 4. A methodaccording to claim 1 wherein said deletion derivative of recombinantcoagulation factor VIII has a molecular weight of 170 kDa to 300 kDa. 5.A method according to claim 1 wherein said recombinant coagulationfactor VIII is a deletion derivative of factor VIII where the whole orpart of the B-domain is missing.
 6. A method according to claim 1wherein said recombinant coagulation factor VIII is factor VIII whichhas been chemically modified by pegylation or covalently linkedcarbohydrates or polypeptides.
 7. A method according to claim 1 whichcomprises subcutaneous administration.
 8. A method according to claim 7wherein the formulation has a volume of 0.1 to 2 ml.
 9. A methodaccording to claim 7 in which the factor VIII activity is from 5000 to100000 IU/ml.
 10. A method according to claim 1 in which the factor VIIIactivity is more than 1500 IU/ml.
 11. A method according to claim 10wherein the formulation has a volume of 0.1 to 2 ml.
 12. A methodaccording to claim 1 in which the deletion derivative factor VIII isdeletion derivative recombinant factor VIII SQ(r-VIII SQ).
 13. A methodaccording to claim 1 wherein the formulation is a stable aqueoussolution ready for administration.
 14. A method according to claim 1wherein the formulation is freeze-dried and reconstituted with sterilewater or a pharmaceutically acceptable buffer solution beforeadministration.
 15. A method according to claim 1 wherein theformulation is mixed or reconstituted with an aqueous solutioncontaining absorption enhancers or protease inhibitors beforeadministration.
 16. Method for treatment of haemophilia by subcutaneous,intramuscular or intradermal administration of pharmaceuticalformulation with a volume of 0.1 to 2 ml, comprising a highly purifiedrecombinant coagulation factor VIII with an activity of at least 1500IU/ml and a non-ionic surfactant, wherein said recombinant coagulationfactor VIII comprises a deletion derivative of factor VIII where thewhole or part of the B-domain is missing; said non-ionic surfactantcomprises at least 0.01 mg/ml of a polyoxyethylene sorbitan fatty acidester; and the formulation further comprises sodium chloride, calciumsalt and an amino acid in an amount of more than 1 mM; which formulationis stabilized without albumin and is capable of increasing the factorVIII activity in the blood to a therapeutic level after subcutaneous,intramuscular or intradermal administration.
 17. The method according toclaim 16 wherein said sodium chloride is present in an amount of morethan 0.1 M, said calcium salt is calcium chloride or calcium gluconateand is present in an amount of more than 0.5 mM, and wherein said aminoacid is L-histidine.
 18. A method according to claim 16 in which thedeletion derivative factor VIII is deletion derivative recombinantfactor VIII SQ (r-VIII SQ).